EP1794180B1 - Peptide cyclisation - Google Patents

Peptide cyclisation Download PDF

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Publication number
EP1794180B1
EP1794180B1 EP05786327A EP05786327A EP1794180B1 EP 1794180 B1 EP1794180 B1 EP 1794180B1 EP 05786327 A EP05786327 A EP 05786327A EP 05786327 A EP05786327 A EP 05786327A EP 1794180 B1 EP1794180 B1 EP 1794180B1
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Prior art keywords
peptide
resin
allyl
protected
group
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EP1794180A1 (en
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Matthieu Giraud
Oleg Werbitzky
Michaela Williner
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Lonza AG
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Lonza AG
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/042General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers characterised by the nature of the carrier
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/665Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • C07K14/68Melanocyte-stimulating hormone [MSH]
    • C07K14/685Alpha-melanotropin
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K7/00Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04Linear peptides containing only normal peptide links
    • C07K7/06Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K7/00Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring

Definitions

  • the present invention relates to a method of synthesis for a cyclic peptide, namely a cyclic peptide comprising ring closure of the carboxy group of the side chain of a one amino acid residue and the amino group of a side chain of a second amino acid residue.
  • Rijkers et al. (An optimized solid phase strategy - including on-resin lactamization- of Astressin, its retro-, inverso- and retro-inverso isomers, 2002, Biopolymers 63, 141-149 ) describe latamisation of Boc-protected 41-mer bound to Rink amide resin. Positions 30 (Glu) and 33 (Lys) are deprotected in a single step by Pd(0) catalyzed removal of allyl and alloc protection groups and are subsequently cyclised by the presence of BOP/HOBt and Hünig base in N-methylpyrrolidone.
  • the peptide was synthesized using a standard orthogonal protection scheme employing Fmoc except for the last residue, which was Boc protected. Subsequently, the N-terminal Boc protection group was removed by acid TFA treatment, simultaneously liberating the peptide from the resin.
  • a disadvantage of this method is that termination of full-length peptide synthesis is a pre-requisite for subsequent cyclisation of a segment of the peptide. Hence the more valuable full-length peptide is subject to yield losses due to unwanted side-reactions (pyroglutamate formation) or incomplete allyl/alloc deprotection. Further, it is not always desirable to use an acid-labile protection group whilst on resin, since this prevents subsequent further derivatisation of the peptide on-resin, e.g. such as N-terminal blocking by acetylation. No reaction may be carried out prior to N-terminal acetylation since this renders the terminal N ⁇ vulnerable to epimerisation at the chiral C ⁇ .
  • Kates et al. (A novel, convenient three dimensional orthogonal strategy for solid-phase synthesis of cyclic peptides, 1993, Tetrahedron Letters 34:1549-1552 ) describes head-to-tail cyclisation of a decameric peptide by side chain anchoring of a C-terminal aspartyl or glutamyl residue to different resin handles which residue is protected at its Ca by an allyl ester protection group. After linear solid phase Fmoc synthesis of the complete peptide chain, the allyl ester moiety is removed by Pd-catalysis, followed by the sequence of N ⁇ -Fmoc removal and subsequent BOP/HOBt/DIEA mediated head-to-tail cyclisation.
  • a limiting disadvantage of this method is that it is tacitly taken into account that partial Fmoc deprotection occurs as a side reaction during the main allyl deprotection step, due to the presence of nucleophilic reagents and because it does not truly affect the reaction scheme. Further completion of Fmoc deprotection takes place subsequently in any event, and is required to allow subsequent head-to-tail peptide bonding. In contrast, cyclisation by means of lactamization of peptide side chain functionalities only is crucially dependent on preserving complete protection of the N ⁇ .
  • Another method of peptide side chain cyclisation is devised according to the present invention, avoiding the disadvantages of the prior art and being particulary useful for cyclisation of peptides comprising aspartyl side chains.
  • a cyclisation method for a peptide comprises the steps of
  • the ' ⁇ -carboxyl group' of an amino acid side chain is understood as being the 'terminal carboxyl group of a side chain irrespective of the carbon chain length
  • the ' ⁇ -amino group' of an amino acid side chain is understood as being the 'terminal' amino group of a side chain irrespective of the carbon chain length.
  • Analogues thereof may well be e.g. side chain isomers thereof.
  • a ⁇ - or ⁇ -amino isomer of lysine is a suitable analogue of natural lysine.
  • the term 'side chain' in respect to an amino acid or amino acid derivative is used in compliance with the respective IUPAC-IUB definition (International Union of Pure and Applied Chemistry and International Union of Biochemistry/Joint Commission on Biochemical Nomenclature, " Nomenclature and Symbolism for Amino Acids and Peptides", Pure Appl. Chem, 56, 595-624 (1984 )).
  • the present reaction will hence be particulary beneficial where multiple glutamyl and/or aspartyl and lysine residues or their analogues such as e.g. nor- or homo-lysine, are present in the final peptide chain whilst only a specific pairing of those residues is scheduled for cyclisation on a firstly synthesized portion of the final full-size peptide chain.
  • This regional-lactamisation approach will be particularly beneficial when the full-size peptide comprises several subsequent subdomains or peptide loop structures for bioactive peptides that are to be stabilized by side-chain cyclisation.
  • Lactamization is a more stable, less redox-vulnerable option than naturally stabilized disulfide-bridging or non-natural, chemical analogues thereof employing amino-acid analogues.
  • the latter functionalities usually prove to be more immunogenic in vivo, whereas the present invention allows use of natural amino acid analogues as a superior option.
  • the peptide contains just one allyl-protected lysine residue and just one allyl-protected aspartyl or glutamyl residue, and hence there is one and only one bonding option upon cyclisation, giving rise to a homogenous product.
  • the peptide according to the method of the present invention can be utilized for further N-terminal peptide elongation, optionally and preferably while on solid phase, be it in the sequential, stepwise mode of adding or modifying amino acid residues or other groups, such as by addition of new, N-protected amino acids or be it by N-terminal condensation reaction with another peptide.
  • the N-terminal residue of the protected peptide having a base-labile protection group at its N ⁇ is said at least one allyl ester-protected aspartyl or glutamyl residue or analogue thereof.
  • N-terminally protected, preferably Fmoc protected, and simultaneously side chain-protected amino acids is that they are prone to a base-catalyzed side reaction upon Fmoc deprotection, giving rise to either aspartimide or glutarimide. It is an undesired shortcoming of present standard solid phase chain elongation synthesis employing FMOC protection groups that is usually believed to be avoided by protection of ⁇ -carboxylic acid groups.
  • the N-terminal allylester-protected acidic residue is an aspartyl residue.
  • Aspartimide formation has a higher reaction rate than glutarimide formation and is usually the more dominant side reaction. It is particularly favored in the dipeptide sequence L-Asp-L-X where X is Gly, Ser, Thr or Asn, and as we report here for the first time, where X is His, easily yielding up to 30 or 40% aspartimide.
  • the Asp-Gly- dipeptide is highly prone to aspartimide formation; usually, this requires use of an additional HMB protection group on the glycine, providing steric hindrance of aspartimide formation as described in Packman et al., 1995, Tetrahedron Lett. 36, 7523 .
  • Gly(HMB) dipeptides is extremly expensive and hence is sought to be avoided. Thus in these instances the present invention offers even a further advantage over the prior art.
  • the allyl and/or alloc deprotection step may be carried out by the methods known in the art, such as e.g. hydrostannolysis with Bu 3 SnH or treatment with tetrakis(triphenylphosphine)palladium(0) [Pd(PPh 3 ) 4 ] in THF at essentially neutral conditions in the presence of a nucleophile such as e.g. morpholine, dimedone, N-methylaniline, HOBt, borohydride or N,N'-dimethylbarbituric acid as an allyl acceptor.
  • a nucleophile such as e.g. morpholine, dimedone, N-methylaniline, HOBt, borohydride or N,N'-dimethylbarbituric acid as an allyl acceptor.
  • Variants of said methods employing different pH exist, but of course care must be paid in view of the pH sensitivity of resin linkage or handle groups and the base sensitive protection group.
  • the allyl deprotection preferably employs catalysis with allyl-group reactive palladium complexes, preferably with Pd(0) complexes, preferably with palladium complexes having C 1 -C 10 trialkyl phosphite, C 3 -C 10 tricycloalkyl phosphite, triarylphosphine or triheteroarylphosphine ligands, wherein said aryl or heteroaryl may be further substituted with electron-donating substitutents or is unsubstituted, more preferably with palladium complexes having phenylphosphine ligands wherein the phenyl may be further substituted with C 1 -C 5 alkyl, preferably wherein the phenyl is tolyl or xylyl, more preferably is phenyl, 2,4-xylyl or o-tolyl.
  • said phosphine ligands are mono-phosphine ligands, more preferably non-chelating, monovalent ligands.
  • the palladium complexes preferably are mono-palladium complexes, the term complex is to be understood as to also comprise di-palladium or higher palladium complexes, though mono-palladium complexes are preferred.
  • methylphenylphosphine ligands and especially the tri(o-tolyl)phosphine ligands improve the catalytic reaction rate, further allowing lowering the total amount of precious metal catalyst used whilst maintaining optimal yields.
  • Pd(0)-catalyzed allyl and allyloxy deprotection compare Jeffrey et al., J. Org. Chem. 1982, 47:587-590 .
  • PdCl 2 (PPh 3 ) 2 / PPh 3 examples of suitable catalyst complexes apart from the strongly preferred Pd(PPh 3 ) 4 are: PdCl 2 (PPh 3 ) 2 / PPh 3 , PdCl 2 (PPh 3 ) 2 / P(o-Tol) 3 , Pd(DBA) 2 / P(o-Tol) 3 or Pd[P(o-Tol) 3 ] 2 ( Organometallics 1995, 14(6):3030-3039 ), Pd(OAc) 2 /triethylphosphite, Pd(OAc) 2 / PPh 3 or Pd(OAc) 2 P(o-Tol) 3 .
  • allyl acceptor reagent or scavenger is any nucleophile such as e.g. morpholine, dimedone, N,N-dimethylbarbituric acid, methylaniline or thiosalicylic acid.
  • Suitable catalytic amounts of Pd(0) complexes preferably are used in an amount of 0.005 eq. to 0.5 eq. catalyst as compared to educt, more preferably are used in an amount of 0.01 up to 0.1 eq. of catalyst, most preferably are used in an amount of 0.015 eq. up to 0.07 eq. of catalyst.
  • reaction temperature is in between 10-60°C, more preferably in between 30-50°C, most preferably at about 40°C.
  • amine-borane complexes are employed in at least 1.5 to 2-fold excess per allyl function of the educt as the nucleophilic allyl group scavenger as described in Gomez-Martin et al., J. Chem. Soc., Perkin Trans. 1(1999): 2871-2874 .
  • high conversion rates along with very short reaction time can be realized, for instance with t-Bu-NH 2 ⁇ BH 3 , Me 2 NH ⁇ BH 3 or NH 3 ⁇ BH 3 .
  • Quaternary amines are excluded from the present definition of suitable complexes, whereas the amine may be preferably a primary or secondary alkyl amine or may be ammonia.
  • said Pd(0) catalyzed allyl-deprotection is carried out as a hydrosilylolysis in the presence of the hydride donor phenyl-trihydrosilane PhSiH 3 or functional derivatives thereof in an aprotic, polar organic solvent such as e.g. dichloromethane, as has been essentially described by Dessolin et al., Tetrahedron Lett. 1995, 36: 5741-5744 .
  • a phenyl-hydrosilane reagent of the generic formula (R1-Ph) n SiH 4-n or PhSiH 3 is employed as the allyl acceptor that is usually in excess of at least 1.5 to 2 eq. wherein R1 is a substituent at the aromatic core and is aryl, alkyl or aralkyl, and n is 1 or 2. Most preferably the allyl acceptor is PhSiH 3 .
  • Both phenylsilanes and suitable amine-borane complexes allow of rapid, complete deprotection in the range of ⁇ 1h, typically at about 20-40 min. Accordingly, they allow mild and short reaction conditions. Other allyl scavengers may require considerable longer reaction times.
  • organic sulfinate R i -SO 2 - may comprise any type of further substituted organic residue, like alkyl, cylcoalkyl, aryl, heteroaryl or aralkyl (cf. Nissan et aL, supra). It may be added in its salt or acid forms.
  • the radical Ri is an optionally further substituted phenyl radical, more preferably is a single or multiple, alkyl-, alkyloxyalkyl- or alkyloxy-substituted phenyl radical, and most preferably is phenyl, xylyl or tolyl, especially p-tolyl.
  • Use of sulfinates also allows use of trialkyl or cycloalkyl phosphite ligand complexes with good yields, in addition to the more preferred triarylic phosphine complexes.
  • the lactamisation reaction is carried out in an essentially similiar manner as standard peptide chain elongation reactions in the presence of base-labile amine-protection groups such as Fmoc, except that the base labile group is carried on the same peptide rather than adding a further N-terminally protected amino acid as in chain elongation.
  • base-labile amine-protection groups such as Fmoc
  • the same chemistry may subsequently be employed for further chain elongation in an optional step d. after cyclisation and deprotection.
  • Both lactamization and chain elongation require a coupling reagent and eventually a coupling additive, depending on the type of primary coupling reagent or auxiliary.
  • Coupling reagents for peptide synthesis are well-known in the art (see Bodansky, M. , Principles of Peptide Synthesis, 2nd ed. Springer Verlag Berlin/Heidelberg, 1993 ; also see discussion of role of coupling additives or auxiliaries therein).
  • Coupling reagents may be anhydrides (e.g. T3P: propanephosphonic anhydride) or other acylating agents such as activated esters or acid halogenides (e.g. ICBF, isobutyl chloroformate), or they may be carbodiimides (e.g.
  • the coupling reagent is selected from the group consisting of uronium salts and phosphonium salts of benzotriazole capable of activating a free carboxylic acid function where the reaction is carried out in the presence of a base.
  • uronium salts and phosphonium salts of benzotriazole capable of activating a free carboxylic acid function where the reaction is carried out in the presence of a base.
  • Suitable and likewise preferred examples of such uronium or phosphonium coupling salts are e.g.
  • HBTU (O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), BOP (benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate), PyBOP (benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate), PyAOP, HCTU (O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronimn hexafluorophosphate), TCTU (O-(1H-6-chlorobenzotriazol-1-yl)-tetramethyluronium hexafluorophosphate), TCTU (O-(1H-6-chlorobenzotriazol-yl)-1,1,3,3-tetramethylumnium tetraflu
  • the base reagent is a weak base whose conjugated acid has a pKa value of from pKa 7.5 to 15, more preferably of from pKa 7.5 to 10, with the exclusion of an ⁇ -amino function of a peptide or amino acid or amino acid derivative, and which base preferably is a tertiary, sterically hindered amine.
  • Hünigbase N,N-diisopropylethylamine
  • N,N-dialkylaniline 2,4,6-triallcylpyridine
  • 2,6-dialkylpyridine or N-alkylmorpholine with the alkyl being straight or branched C 1 -C 4 alkyl, more preferably it is N-methylmorpholine or collidine (2,4,6-trimethylpyridine), most preferably it is collidine.
  • coupling additives in particular of coupling additives of the benzotriazole type, is also known (see Bodansky, supra). Their use is particularly preferred when employing highly activating uronium or phosphonium salt coupling reagents.
  • the coupling reagent additive is a nucleophilic hydroxy compound capable of forming activated esters, more preferably having an acidic, nucleophilic N-hydroxy function wherein N is imide or is N-acyl or N-aryl substituted triazeno, most preferably the coupling additive is a N-hydroxybenzotriazole derivative (or 1-hydroxybenzotriazole derivative) or is an N-hydroxybenzotriazine derivative.
  • N-hydroxy compounds have been described in WO 94/07910 and EP-410 182 and the respective disclosure is incorporated by reference hereto. Examples are e.g. N-hydroxysuccinimide, N-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HOOBt), 1-hydroxy-7-azabenzotriazole (HOAt) and N-hydroxybenzotriazole (HOHt).
  • N-hydroxybenzotriazine derivatives are particularly preferred, in a most preferred embodiment, the coupling reagent additive is N-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine.
  • Ammonium salt compounds of coupling additives are known and their use in coupling chemistry has been described, for instance in US 4,806,641 .
  • the uronium or phosphonium salt coupling reagent is an uronium salt reagent and preferably is HCTU, TCTU or HBTU and even more preferably is used in the reaction in combination with N-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine or a salt thereof.
  • This embodiment is mainly preferred for use in chain elongation step of peptide synthesis after removal of the base-labile N ⁇ -protection group, but may as well be used for lactamization reaction during side-chain cyclisation.
  • HCTU and TCTU are defined as to be encompassed by the term 'uronium salt reagent' despite that these compounds and possible analogues have been shown to comprise an isonitroso moiety rather than an uronium moiety by means of crystal structure analysis ( O. Marder, Y. Shvo, and F. Albericio "HCTU and TCTU: New Coupling Reagents: Development and Industrial Applications", Poster, Presentation Gordon Conference February 2002 ), an N-amidino substituent on the heterocyclic core giving rise to a guanidium structure instead.
  • such class of compounds is termed the 'guanidium-type subclass' of uronium salt reagents according to the present invention.
  • the coupling reagent is a phosphonium salt of the benzotriazol such as e.g. BOP, PyBOP or PyAOP.
  • Deprotection of the base labile N ⁇ may be carried out as routinely done in the art, e.g. with 20% piperidine in N-methylmorpholine.
  • the resin or resin handle composite entity may in principle be any resin employed for synthesis, such as for example a polystyrene-divinylbenzene resin as used by Merrifield along with hydroxybenzyl-phenyl integral linker moieties or by Wang with hydroxybenzyl-p-benzyloxy moieties, such as for example moieties to which e.g. more acid-labile linkers may be further grafted, or alternatively the latter linkers may be integrally or directly linked to the resin.
  • a solid phase resin for use in synthesis necessarily comprises at least an integral linker or handle which is part of the solid phase core material; such linker or handle may be considered as an immobilized protection group ( Guillier et al., Chem. Rev.
  • Examples are e.g. Sieber resin, related xanthenyl type PAL handle resins, Rink amide resin, Rink acid resin, more complex PEG-grafted polystyrene resins such as tentagel-based Novasyn TG (Novabiochem, Merck Biosciences, Germany) which are available with different grafted handles such as 2-chlorotrityl, or resins that are constituted by grafting functional handles onto matrix material such as silica gels.
  • the resin is a trityl resin or resin handle, such resin is a 4-methoxy- or 4,4'-dimethoxytrityl resin.
  • Resins as used in the present invention are of standard mesh size, which is about 50-500 mesh, more preferably 100 to 400 mesh.
  • a resin or solid-phase R"' as shown in formula IV is to be construed as to comprise a crosslinked, polymeric matrix material which may be bound to the handle moiety specified in formulas IV to VII by way of any kind of chemically inert alkyl, alkyloxy, aryloxy or alkyl ester spacer or linker which is to be considered an integral part of R"'.
  • the chemical nature of the resin material and in particular the chemical nature of the handle group may well influence synthetic efficiency of coupling and especially lactamisation reactions in a yet poorly understood fashion.
  • the resin or resin handle is of formula IV as set forth in the claims in detail, more preferably of formula VI and most preferably of formula VII as set forth in the claims in detail.
  • examples of such resins or resin handles are (4-methoxyphenyl)-methyl- and (4-methylphenyl)-methyl-polystyrene ( Atkinson et al., 2000, J. Org. Chem. 65, 5048 ), resins in O- or N-linkage to the peptide moiety and their PEG-resin derivatives, respectively. Further examples are e.g.
  • HMPB-MBHA or HWPB-BHA resin Sieber et al., 1987, Tetrahedron Lett. 28, 6147
  • acid-labile Rink amide resin or Rink acid resin Rink et al., 1987, Tetrahedron Lett. 28, 3787 .
  • the term 'acid-labile' refers to essentially quantitative cleavage in 2-10% TFA in dichloromethane at ambient temperature for at least an hour.
  • radical A (as given e.g. in formula II or III) comprises a resin handle or resin linkage moiety with the exception of resin handles comprising an allyloxycarbonyl moiety. More preferably, such resin or resin handle is of formula IV wherein R"' is a resin, and R1", R2", R3" are, independently, hydrogen, C 1 -C 4 alkyl or C 1 -C 4 alkoxy, and may be the same or different with the proviso that only one of R1", R2" may be hydrogen, and wherein L is oxygen, sulfur, nitrogen or is of formula V
  • resin or resin handle is of formula VI, the above definitions for radicals R"', R1" and R2" applying with the proviso that L is selected from the group consisting of oxygen and nitrogen,
  • the resin or resin handle is of formula VII, the above definitions for radicals R"', R1" and R2" applying with the proviso that L is selected from the group consisting of oxygen and nitrogen,
  • R1", R2" are, where L is oxygen, independently hydrogen, methyl or methoxy with the proviso that only one of R1", R2" may be hydrogen, and where L is nitrogen, independently are methyl or methoxy, preferably are methoxy. Even more preferably then, L is oxygen, R1" is hydrogen and R2" is methyl or methoxy. Most preferably, R2" is methyl.
  • the peptide sequence according to the present invention is Ac-Nle-cyclo(Asp-His-D-Phe-Arg-Trp-Lys) or NIe-cyclo(Asp-His-D-Phe-Arg-Trp-Lys), a lactam bond being in place between the Asp and Lys side chains as shown in scheme 1.
  • Said peptide is a pharmaceutically active melanocortin receptor-specific peptide useful for treatment of sexual dysfunction, including male erectile dysfunction and female sexual dysfunction in humans.
  • the peptide sequence according to the present invention consists of or comprises at least the partial sequence cyclo(Asp-His-Phe.Arg-Trp-Lys), wherein the Phe residue may also be D-Phe, or the respective D-or L-isomer of Phe(4-7), Phe(4-Br), Phe(4-CF 3 ), Phe(4-CI), Phe(2,4-diCl), Phe(3,4-diCl), Phe(3,4-diF), Phe(4-I), Phe(3,4-di-OMe), Phe(4-Me) or Phe(4-NO 2 ).
  • the Arg may also be D-Arg, or the respective D- or L-isomer of Arg(NO 2 ), Arg(Tos), Arg(Pbf), Arg(Mtr), Arg(Me) or Arg(Pmc).
  • the arginine side chain may be preferably covalently protected during synthesis e.g. with tosyl (Tos), benzyloxycarbonyl, pentamethylchromanesulfonyl (Pmc), pentamethyldihydrobenzofuranesulfonyl (Pbf), 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr) and its 4 tBu-2,3,5,6-tetramethyl homologue (Tart), adamantyloxycarbonyl or Boc.
  • Tos tosyl
  • Pmc pentamethylchromanesulfonyl
  • Pbf pentamethyldihydrobenzofuranesulfonyl
  • Mtr 4-methoxy-2,3,6-trimethylbenzenesulfonyl
  • Tart 4-methoxy-2,3,6-trimethylbenzenesulfonyl
  • Tart adamantyloxycarbonyl or Boc
  • Trp is preferably protected during synthesis with Boc. Optionally, it may be N-protected with formyl or mesitylenesulfonyl.
  • His is preferably protected by N-trityl protection group.
  • N-trityl protection group may be likewise N-protected with Boc, methyltrityl or tosyl.
  • Fmoc deprotection was carried out with 20% piperidine in NMP. Subsequently, chain elongation was carried out for 30 min. in DMF at about room temperature (40°C) for 1 h with Fmoc-L-Nle (leq.) in the presence of 1 eq. HCTU and 3 eq. HOOBt, 3 eq. DIEA. After filtering off the resin-bound product and washing with DMF, the Fmoc moiety on Nle was removed with 20% piperidine in NMP, and an N-terminal acetyl group incorporated by incubation in pyridine with about 1.5 eq. of acetic anhydride for 1-2 h at room temperature.
  • Reaction 2.1 was repeated, with the following modifications: Instead of 0.1 eq. of Pd(PPh 3 ) 4 , 0.05 eq. of Pd(OAc) 2 in the presence of 0.05 eq. of tri(toly)phosphine were used. Further, 2.2 eq. of sodium p-toluenesulfinate were added as scavenger. Even after 2 hours, conversion had only taken place in trace amounts; raising the temperature to 60°C did not change that.
  • Catalyst Pd(P[o-Tol] 3 ) 2 was obtained as described in Paul et al., Organometallics 1995, 14(6), 3030-3039 . Catalyst was solubilized in AcOH/DMF(2:1). The reaction was carried out as described in chapter 2.1, but with using 0.05 eq. of Pd(P[o-Tol] 3 ) 2 and 2.2 eq. of sodium p-toluenesulfinate were added as scavenger under steady nitrogen bubbling. Reaction took place for 30 min. at 40°C.
  • Reaction 4.1 was repeated as described above, except that now 0.1 eq. of Pd(PPh 3 ) 4 were used as the catalyst and 30-60 min. reaction time was employed for allyl/alloc deprotection. The yield of cleaved, acetylated mature peptide amounted to 82%.

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  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
EP05786327A 2004-09-20 2005-09-20 Peptide cyclisation Revoked EP1794180B1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
PL05786327T PL1794180T3 (pl) 2004-09-20 2005-09-20 Cyklizacja peptydu
EP05786327A EP1794180B1 (en) 2004-09-20 2005-09-20 Peptide cyclisation
SI200530338T SI1794180T1 (sl) 2004-09-20 2005-09-20 Peptidna ciklizacija

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP04022310 2004-09-20
EP05014954 2005-07-11
PCT/EP2005/010133 WO2006032457A1 (en) 2004-09-20 2005-09-20 Peptide cyclisation
EP05786327A EP1794180B1 (en) 2004-09-20 2005-09-20 Peptide cyclisation

Publications (2)

Publication Number Publication Date
EP1794180A1 EP1794180A1 (en) 2007-06-13
EP1794180B1 true EP1794180B1 (en) 2008-06-04

Family

ID=35445710

Family Applications (1)

Application Number Title Priority Date Filing Date
EP05786327A Revoked EP1794180B1 (en) 2004-09-20 2005-09-20 Peptide cyclisation

Country Status (22)

Country Link
US (1) US20080171849A1 (sl)
EP (1) EP1794180B1 (sl)
JP (1) JP2008513403A (sl)
KR (1) KR20070085247A (sl)
AP (1) AP2007003958A0 (sl)
AT (1) ATE397618T1 (sl)
AU (1) AU2005287578A1 (sl)
BR (1) BRPI0515490A (sl)
CA (1) CA2581215A1 (sl)
DE (1) DE602005007364D1 (sl)
DK (1) DK1794180T3 (sl)
EA (1) EA010786B1 (sl)
ES (1) ES2308539T3 (sl)
IL (1) IL182024A0 (sl)
MX (1) MX2007003229A (sl)
NO (1) NO20071927L (sl)
PL (1) PL1794180T3 (sl)
PT (1) PT1794180E (sl)
SI (1) SI1794180T1 (sl)
TN (1) TNSN07099A1 (sl)
TW (1) TW200626611A (sl)
WO (1) WO2006032457A1 (sl)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010514728A (ja) * 2006-12-29 2010-05-06 エフ.ホフマン−ラ ロシュ アーゲー 環状ペプチドの合成方法
JP2011524340A (ja) 2008-05-21 2011-09-01 ニュー・ワールド・ラボラトリーズ・インコーポレイテッド 選択的カスパーゼ阻害剤およびその使用
MX2010013436A (es) 2008-06-09 2011-06-21 Palatin Technologies Inc Peptidos especificos del receptor de melanocortina para el tratamiento de disfuncion sexual.
UY32690A (es) 2009-06-08 2011-01-31 Astrazeneca Ab Péptidos específicos para receptores de melanocortina
RU2012125033A (ru) * 2009-11-16 2014-01-20 Ипсен Фарма С.А.С. СПОСОБ СИНТЕЗА Ас-Arg-ЦИКЛО(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-NH2
CN102725305B (zh) 2009-11-23 2016-08-24 帕拉丁科技公司 黑皮质素-1受体特异性环肽
EP2504351A4 (en) 2009-11-23 2013-10-30 Palatin Technologies Inc MELANOCORTIN-1 RECEPTOR-SPECIFIC LINEAR PEPTIDE
US9944674B2 (en) 2011-04-15 2018-04-17 Genesis Technologies Limited Selective cysteine protease inhibitors and uses thereof
EP3947411A4 (en) * 2019-04-04 2022-11-09 Versitech Limited CYCLIC COMPOUNDS AND METHODS OF MAKING AND USING THEIR
WO2021148594A1 (en) * 2020-01-23 2021-07-29 Nosopharm Chemical synthesis of the peptidic part of bioactive natural products
CN111777669B (zh) * 2020-07-28 2022-03-08 安徽工程大学 一种抗肿瘤活性肽、合成方法及应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CZ20003798A3 (cs) * 1998-04-15 2001-08-15 Aventis Pharmaceuticals Products, Inc. Způsob výroby cyklických peptidů vázaných k polymernímu nosiči
US6579968B1 (en) * 1999-06-29 2003-06-17 Palatin Technologies, Inc. Compositions and methods for treatment of sexual dysfunction

Also Published As

Publication number Publication date
EP1794180A1 (en) 2007-06-13
PT1794180E (pt) 2008-08-22
AP2007003958A0 (en) 2007-07-30
DK1794180T3 (da) 2008-09-15
EA010786B1 (ru) 2008-10-30
EA200700659A1 (ru) 2007-10-26
SI1794180T1 (sl) 2008-10-31
US20080171849A1 (en) 2008-07-17
WO2006032457A9 (en) 2006-05-26
DE602005007364D1 (de) 2008-07-17
ES2308539T3 (es) 2008-12-01
IL182024A0 (en) 2007-07-24
TNSN07099A1 (en) 2008-06-02
JP2008513403A (ja) 2008-05-01
AU2005287578A1 (en) 2006-03-30
KR20070085247A (ko) 2007-08-27
MX2007003229A (es) 2007-06-05
CA2581215A1 (en) 2006-03-30
WO2006032457A1 (en) 2006-03-30
BRPI0515490A (pt) 2008-07-29
PL1794180T3 (pl) 2008-10-31
NO20071927L (no) 2007-06-18
ATE397618T1 (de) 2008-06-15
TW200626611A (en) 2006-08-01

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